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Abstract:

An in vitro method for screening candidate compounds for the preventive or
curative treatment of acne, includes the determination of the capacity of
a compound to modulate the expression or the activity of ELOVL5 and the
use of modulators of the expression or activity of this enzyme for the
treatment of acne or skin disorders associated with a hyperseborrhea;
methods for the in vitro diagnosis or prognosis of these pathologies are
also described.

Claims:

1. An in vitro method for screening candidate compounds for the preventive
and/or curative treatment of acne, and/or skin disorders associated with
a hyperseborrhea, comprising determining the capacity of a candidate
compound to modulate the expression or activity of ELOVL5 or the
expression of its gene or the activity of at least one of its promoters.

2. An in vitro method for screening candidate compounds for the preventive
and/or curative treatment of acne and/or skin disorders associated with a
hyperseborrhea as defined by claim 1, comprising the following steps:a.
preparing at least two biological samples or reaction mixtures;b.
bringing one of the samples or reaction mixtures into contact with one or
more test compounds;c. measuring the expression or activity of the
protein ELOVL5, the expression of its gene or the activity of at least
one of its promoters, in biological samples or reaction mixtures;d.
selecting the compounds for which a modulation of the expression or
activity of the protein ELOVL5 or a modulation of the expression of its
gene or a modulation of the activity of at least one of its promoters, is
measured in the sample or mixture treated in b), compared with the
untreated sample or mixture.

3. The in vitro method as defined by claim 2, wherein the compounds
selected in step d) inhibit the expression or the activity of the protein
ELOVL5, or the expression of its gene or the activity of at least one of
its promoters.

4. The in vitro method as defined by claim 2, wherein the biological
samples are cells transfected with a reporter gene that is operably
linked to all or part of the promoter of the gene encoding the protein
ELOVL5, and in that step c) comprises measuring the expression of the
said reporter gene.

5. The in vitro method as defined by claim 2, wherein the biological
samples are cells expressing the gene encoding the protein ELOVL5, and in
that step c) comprises measuring the expression of the said gene.

6. The in vitro method as defined by claim 4, in which the cells are
sebocytes.

7. The in vitro method as defined by claim 4, in which the cells are cells
transformed with a heterologous nucleic acid encoding ELOVL5.

8. The in vitro method as defined by claim 2, in which the expression of
the gene is determined by measuring the level of transcription of the
said gene.

9. The in vitro method as defined by claim 2, in which the expression of
the gene is determined by measuring the level of translation of the said
gene.

10. The in vitro method as defined by claim 2, wherein step a) comprises
preparing reaction mixtures each comprising an enzyme ELOVL5 and a
substrate of the enzyme, and in that step c) comprises measuring the
enzyme activity.

Description:

CROSS-REFERENCE TO PRIORITY/PCT APPLICATIONS

[0001]This application claims priority under 35 U.S.C. § 119 of FR
0653032, filed Jul. 19, 2006, and is a continuation/national phase of
PCT/FR 2007/051685, filed Jul. 18, 2007, and designating the United
States (published in the French language on Jan. 24, 2008 as WO
2008/009858 A2; the title and abstract were also published in English),
each hereby expressly incorporated by reference in its entirety and each
assigned to the assignee hereof.

BACKGROUND OF THE INVENTION

[0002]1. Technical Field of the Invention

[0003]The present invention relates to the identification and
administration of ELOVL5 modulating compounds for the treatment of acne
and skin disorders associated with a hyperseborrhea. This invention also
relates to methods for the in vitro diagnosis or in vitro prognosis of
these pathologies.

[0004]2. Description of Background and/or Related and/or Prior Art

[0005]A hyperseborrheic greasy skin is characterized by excessive
secretion and excretion of sebum. Conventionally, a sebum level greater
than 200 μg/cm2, measured in the region of the forehead, is
considered as being characteristic of a greasy skin. A greasy skin is
often associated with a desquamation defect, a glistening complexion and
a thick skin grain. In addition to these aesthetic disorders, excess
sebum can serve as a support for the anarchical development of
saprophytic bacterial flora (P. acnes in particular), and cause the
appearance of comedones and/or acne lesions.

[0006]This stimulation of the production of sebaceous glands is induced by
androgens. Acne is in fact a chronic disease of the pilosebaceous
follicle under hormonal control. A hormone therapy against acne is one
possibility of treatment for women, the aim being to prevent the effects
of androgens on the sebaceous gland. In this context, use is generally
made of oestrogens, anti-androgens or agents reducing the production of
androgens by the ovaries or the adrenal gland. The anti-androgens
administered for the treatment of acne include in particular
spironolactone, cyproterone acetate and flutamide. However, these agents
have potentially severe side effects. Thus, any pregnancy must be
absolutely prevented, in particular because of a risk of feminization for
the male foetus. These agents are banned in male patients.

[0007]Need therefore exists to identify mediators downstream of the action
of the steroid hormones and to modulate them in order to provide a
similar therapeutic profile, but with reduced side effects.

SUMMARY OF THE INVENTION

[0008]It has now been discovered that the ELOVL5 gene encoding a fatty
acyl CoA elongase was expressed in the human sebaceous glands, and that
its expression was regulated by androgens, in vivo, in a mouse preputial
gland model. Thus, targeting the ELOVL5 gene or its expression product is
now proposed to prevent and/or improve acne and skin disorders associated
with a hyperseborrhea, in particular the appearance of greasy skin.

[0009]The expression acne means all the forms of acne, namely, in
particular acne vulgaris, comedo type acne, polymorphic acne,
nodulocystic acne, acne conglobata, or secondary acnes such as solar
acne, acne medicamentosa or occupational acne. This invention also
provides in vitro diagnostic or in vitro prognostic methods based on the
detection of the level of expression or activity of the enzyme ELOVL5.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIGS. 1A, 1B and 1C show the expression of ELOVL5 in the sebaceous
gland of the mouse skin and in the mouse preputial gland by in situ
hybridization,

[0011]FIGS. 2A, 2B and 2C show the expression of ELOVL5 in the preputial
gland of mice by in situ hybridization,

[0012]FIGS. 3A and 3B are graphs which show the measurement of the
expression of the ELOVL5 gene in gonadectomized male mice treated with
various vehicles.

DETAILED DESCRIPTION OF BEST MODE AND SPECIFIC/PREFERRED EMBODIMENTS OF
THE INVENTION

ELOVL5

[0013]The enzyme ELOVL5 belongs to the family of ELOVL (for "Elongation of
very long chain fatty acids") enzymes which comprises 5 members
identified to date in humans. Recently, the gene encoding the enzyme
ELOVL3 has been studied and it has been shown that the product of
expression of the ELOVL3 gene (mRNA) was found more particularly in the
sebaceous glands of the skin and the epithelial cells of the hair
follicles (Westerberg et al., J. Bio. Chem., 2004, 279, vol. 7,
5621-5629). ELOVL3 exhibits nevertheless no significant sequence homology
with ELOVL5. It is therefore surprisingly that the inventors identified
ELOVL5, and not so much ELOVL3, to be involved in acne phenomena.

[0014]In the context of the present invention, the term "ELOVL5 gene" or
"ELOVL5 nucleic acid" means the gene or nucleic acid sequence which
encodes ELOVL5. If the intended target is preferably the human gene or
its expression product, the invention may also call into play cells
expressing a heterologous ELOVL5, through genomic integration or
transient expression of an exogenous nucleic acid encoding the enzyme.

[0015]A human cDNA sequence for ELOVL5 is reproduced in the annex (SEQ ID
No. 1). It is the sequence NM021814.3 whose coding moiety is located from
nucleic acid 337 to 1236.

Diagnostic Applications

[0016]The present invention features an in vitro method for the diagnosis
or monitoring of the progression of acne lesions or of a skin disorder
associated with a hyperseborrhea in a subject, comprising comparing the
expression or the activity of the protein ELOVL5, the expression of its
gene or the activity of at least one of its promoters, in a biological
sample from a subject compared with a biological sample from a control
subject.

[0017]The expression of the protein may be determined by an assay of this
protein by radioimmunoassay, for example by ELISA assay. Another method,
in particular for measuring the expression of the ELOVL5 gene, is to
measure the quantity of corresponding mRNA, by any method as described
above. An assay of the activity of the ELOVL5 protein may also be
employed.

[0018]In the context of a diagnosis, the "control" subject is a "healthy"
subject.

[0019]In the context of a monitoring of the progression of acne lesions or
of a skin disorder linked to a hyperseborrhea, the "control subject"
refers to the same subject at a different time, which preferably
corresponds to the start of the treatment (To). This measurement of the
difference in the expression or the activity of ELOVL5 makes it possible
in particular to monitor the efficacy of a treatment, in particular a
treatment with an ELOVL5 modulator, as indicated above, or with another
treatment against acne or a skin disorder associated with a
hyperseborrhea. Such a monitoring can reassure the patient regarding the
justification or the need for pursuing this treatment.

[0020]The present invention also features an in vitro method for
determining the predisposition of a subject to develop acne lesions or a
skin disorder associated with a hyperseborrhea, comprising comparing the
expression or the activity of the protein ELOVL5, the expression of its
gene or the activity of at least one of its promoters, in a biological
sample from a subject compared with a biological sample from a control
subject.

[0021]Here again, the expression may be determined by an assay of the
ELOVL5 protein by radioimmunoassay, for example by ELISA assay. Another
method, in particular for measuring the expression of the ELOVL5 gene, is
to measure the quantity of corresponding mRNA by any method as described
above. An assay of the activity of ELOVL5 may also be employed.

[0022]The subject tested is here an asymptomatic subject with no skin
disorder linked to a hyperseborrhea or an acne. The "control" subject in
this method means a "healthy" reference subject or population. The
detection of this predisposition allows the putting in place of a
preventive treatment and/or an increased monitoring of the signs linked
to acne or to a skin disorder associated with a hyperseborrhea.

[0023]In these in vitro diagnostic or prognostic methods, the biological
test sample may be any biological fluid sample or a sample of a biopsy.
Preferably, the sample may be a preparation of skin cells obtained for
example by desquamation or biopsy. It may also be sebum.

Screening Methods

[0024]This invention also features an in vitro method for screening
candidate compounds for the preventive and/or curative treatment of acne,
and/or of the skin disorders associated with a hyperseborrhea, comprising
determining the capacity of a compound to modulate the expression or
activity of ELOVL5 or the expression of its gene or the activity of at
least one of its promoters, the said modulation indicating the usefulness
of the compound for the preventive or curative treatment of acne or of
the skin disorders associated with a hyperseborrhea. The method therefore
makes it possible to select the compounds capable of modulating the
expression or activity of ELOVL5, or the expression of its gene or the
activity of at least one of its promoters.

[0025]More particularly, this invention features an in vitro method for
screening candidate compounds for the preventive and/or curative
treatment of acne and/or of the skin disorders associated with a
hyperseborrhea, comprising the following steps:

[0026]a. preparing at least two biological samples or reaction mixtures;

[0027]b. bringing one of the samples or reaction mixtures into contact
with one or more test compounds;

[0028]c. measuring the expression or activity of the protein ELOVL5, the
expression of its gene or the activity of at least one of its promoters,
in biological samples or reaction mixtures;

[0029]d. selecting the compounds for which a modulation of the expression
or activity of the protein ELOVL5, the expression of its gene or the
activity of at least one of its promoters, is measured in the sample or
mixture treated in b), compared with the untreated sample or mixture.

[0030]The expression "modulation" means any effect on the expression or
activity of the enzyme, on the expression of its gene or on the activity
of at least one of its promoters, namely, optionally a partial or
complete stimulation, but preferably a partial or complete inhibition.
Thus, the compounds tested in step d) above preferably inhibit the
expression or activity of the protein ELOVL5, the expression of its gene
or the activity of at least one of its promoters. The difference in
expression obtained with the test compound compared with a control
prepared in the absence of the compound is significant from 25% or more.

[0031]In the present text, unless otherwise specified, "expression of a
protein" means the quantity of this protein.

[0032]The expression "activity of a protein" means its biological
activity.

[0033]The expression "activity of a promoter" means the capacity of this
promoter to trigger the transcription of the DNA sequence coded
downstream of this promoter (and therefore indirectly the synthesis of
the corresponding protein).

[0034]The test compounds may be of any type. They may be of a natural
origin or may have been produced by chemical synthesis. This may be a
library of structurally defined chemical compounds, non-characterized
compounds or substances or a mixture of compounds.

[0035]Various techniques may be used to test these compounds and identify
the compounds of therapeutic interest, modulators of the expression or
the activity of ELOVL5.

[0036]According to a first embodiment, the biological samples are cells
transfected with a reporter gene that is operably linked to all or part
of the promoter of the ELOVL5 gene, and step c) described above entails
measuring the expression of the said reporter gene.

[0037]The reporter gene may in particular encode an enzyme which, in the
presence of a given substrate, leads to the formation of colored
products, such as CAT (chloramphenicol acetyltransferase), GAL
(beta-galactosidase) or GUS (beta-glucuronidase). This may also be the
luciferase gene or GFP (Green Fluorescent Protein). The assay of the
protein encoded by the reporter gene, or its activity, is carried out in
a conventional manner by calorimetric, fluorometric or chemiluminescent
techniques, among others.

[0038]According to a second embodiment, the biological samples are cells
expressing the gene encoding ELOVL5, and step c) described above entails
measuring the expression of the said gene.

[0039]The cell employed here may be of any type. This may be a cell
endogenously expressing the ELOVL5 gene, such as, for example, a liver
cell, an ovarian cell or even better a sebocyte. It is also possible to
employ organs of human or animal origin, such as for example the
preputial gland, clitorial gland or sebaceous gland of the skin.

[0040]This may also be a cell transformed with a heterologous nucleic acid
encoding ELOVL5, preferably of human origin, or of mammalian origin.

[0041]A wide variety of host cell systems may be employed, such as, for
example, Cos-7, CHO, BHK, 3T3, HEK293 cells. The nucleic acid may be
stably or transiently transfected by any method known to one skilled in
this art, for example using calcium phosphate, DEAE-dextran, liposome,
viruses, electroporation or microinjection.

[0042]In these methods, the expression of the ELOVL5 gene may be
determined by measuring the level of transcription of the said gene, or
its level of translation.

[0043]The expression level of transcription of a gene means the quantity
of corresponding mRNA produced. The expression level of translation of a
gene means the quantity of corresponding protein produced.

[0044]One skilled in the art is familiar with techniques allowing the
quantitative or semi-quantitative detection of the mRNA of a gene of
interest. The techniques based on the hybridization of mRNA with specific
nucleotide probes are the most common (Northern Blot, RT-PCR, protection
using RNase). It may be advantageous to employ detection markers such as
fluorescent, radioactive or enzymatic agents or other ligands (for
example avidin/biotin).

[0045]In particular, the expression of the gene may be measured by
real-time PCR or by protection using RNase. The expression protection
using RNase means the detection of a known mRNA among poly(A) RNAs of a
tissue, which may be carried out with the aid of a specific hybridization
with a labeled probe. The probe is a labeled (radioactive) complementary
RNA for the messenger to be detected. It may be constructed from a known
mRNA whose cDNA, after RT-PCR, has been cloned into a phage. The poly(A)
RNA of the tissue where the sequence is to be detected is incubated with
this probe under slow hybridization conditions in liquid medium. RNA:RNA
hybrids are formed between the mRNA to be detected and the anti-sense
probe. The hybridized medium is then incubated with a mixture of
ribonucleases specific for single-stranded RNA, such that only the
hybrids formed with the probe can withstand this digestion. The product
of digestion is then deproteinized and repurified before being analyzed
by electrophoresis. The labeled hybridized RNAs are detected by auto
radiography.

[0046]The level of translation of the gene is evaluated for example by
immunological assay of the product of the said gene. The antibodies
employed for this effect may be of the polyclonal or monoclonal type.
Their production involves conventional techniques. An anti-ELOVL5
polyclonal antibody may, inter alia, be obtained by immunization of an
animal such as a rabbit or a mouse, with the whole enzyme. The anti-serum
is collected and then depleted according to methods known per se by
persons skilled in the art. A monoclonal antibody may, inter alia, be
obtained by the conventional Kohler and Milstein method (Nature (London),
256: 495-497 (1975)). Other methods of preparation of monoclonal
antibodies are also known. It is possible, for example, to produce
monoclonal antibodies by expressing a nucleic acid cloned from a
hybridoma. It is also possible to produce antibodies by the phage display
technique by introducing antibody cDNAs into vectors, which are typically
filamentous phages which display V gene libraries at the surface of the
phage (for example, fUSE5 for E. coil).

[0047]The immunological assay may be carried out in a solid phase or in a
homogeneous phase; in a single stage or in two stages; as a sandwich
method or as a competitive method, by way of non-limiting examples.
According to a preferred embodiment, the capture antibody is immobilized
on a solid phase. It is possible to employ, by way of non-limiting
examples of a solid phase, microplates, in particular polystyrene
microplates, or solid particles or beads, paramagnetic beads.

[0048]ELISA assays, radio-immunoassays or any other detection technique
may be carried out in order to reveal the presence of the
antigen-antibody complexes formed.

[0049]The characterization of the antigen-antibody complexes, and more
generally of the isolated or purified proteins, but also recombinant
proteins (obtained in vitro and in vivo), may be carried out by mass
spectrometry analysis. This identification is made possible by virtue of
the analysis (determination of the mass) of peptides generated by the
enzymatic hydrolysis of the proteins (trypsin in general). Generally, the
proteins are isolated according to methods known to one skilled in this
art, prior to the enzymatic digestion. The analysis of the peptides (in
hydrolysate form) is performed by separation of the peptides by HPLC
(nano-HPLC) based on their physicochemical properties (reversed phase).
The determination of the mass of the peptides thus separated is carried
out by ionization of the peptides or by direct coupling to mass
spectrometry (electrospray ESI mode), or after deposition and
crystallization in the presence of a matrix known to one skilled in this
art (analysis in MALDI mode). The proteins are then identified using
appropriate software (for example Mascot).

[0050]According to a third embodiment, step a) described above entails
preparing reaction mixtures each comprising an enzyme ELOVL5 and a
substrate of the enzyme, and step c) described above entails measuring
the enzyme activity.

[0051]The enzyme ELOVL5 may be produced according to customary techniques
using Cos-7, CHO, BHK, 3T3 and HEK293 cells. It may also be produced with
the aid of microorganisms such as bacteria (for example, E. coli or B.
subtilis), yeasts (for example Saccharomyces, Pichia) or insect cells,
such as Sf9 or Sf21.

[0052]The determination of the enzymatic activity preferably comprises the
determination of the synthetase activity via the use of radioactive
precursors and the synthesis of radioactive products.

[0054]By way of example, the enzyme ELOVL5 is incubated in a reaction
medium (0.25 ml total) containing 100 μM of Tris-HCl, pH 7.4, 60 μM
of palmitoyl CoA, 500 μM NADPH and 30 μg of enzyme. After 2 minutes
of preincubation at 37° C., the reaction is initiated by the
addition of 60 μM of malonyl-CoA (containing 0.037 μCi of
[2-14C]malonyl-CoA) and left for 5 minutes at 37° C. The
incubation is stopped by the addition of 0.5 ml of 15% KOH methanol and
saponified at 65° C. for 45 minutes. The samples are then cooled
and acidified with 0.5 ml of ice-cold 5 N HCl. The free fatty acids are
extracted from the mixture three times with 1 ml of hexane. The fractions
extracted with hexane are dried under vacuum, and after addition of 3 ml
of scintillant, the radioactivity incorporated is counted. The controls
are performed in parallel by incubation without the enzyme.

Modulators of the Enzyme

[0055]The present invention also features the use of a modulator of the
human enzyme ELOVL5 which can be obtained by one of the above methods,
for the preparation of a medicament useful for the preventive and/or
curative treatment of acne, and/or of skin disorders associated with a
hyperseborrhea.

[0056]A method for the preventive and/or curative treatment of acne, or of
skin disorders associated with a hyperseborrhea, is thus described here,
the regime or regimen comprising the administration of a therapeutically
effective quantity of a modulator of the human enzyme ELOVL5, to a
patient requiring such a treatment.

[0057]This invention also features the cosmetic administration of a
modulator of the human enzyme ELOVL5 for the aesthetic treatment of
greasy skins.

[0058]Preferably, the modulator is an inhibitor of the enzyme. The term
"inhibitor" refers to a chemical compound or substance which
substantially eliminates or reduces the enzymatic activity of ELOVL5. The
term "substantially" means a reduction of at least 25%, preferably of at
least 35%, preferably still of at least 50%, and more preferably of at
least 70% or 90%. More particularly, it may be a compound which interacts
with, and blocks, the catalytic site of the enzyme (irreversibly or
otherwise), such as compounds of the competitive inhibitor type.

[0059]A preferred inhibitor interacts with the enzyme in solution at
inhibitor concentrations of less than 1 μM, preferably of less than
0.1 μM, preferably still of less than 0.01 μM.

[0060]The modulator compound may be an anti-ELOVL5 inhibitory antibody,
preferably a monoclonal antibody. Advantageously, such an inhibitory
antibody is administered in a quantity sufficient to obtain a plasma
concentration of about 0.01 μg per ml to about 100 μg/ml,
preferably of about 1 μg per ml to about 5 μg/ml.

[0061]The modulator compound may also be a polypeptide, a DNA or RNA
anti-sense polynucleotide, an si-RNA or a PNA ("peptide nucleic acid",
polypeptide chain substituted with purine and pyrimidine bases whose
spatial structure mimics that of DNA and allows hybridization thereto).

[0062]The modulator compounds are formulated in a pharmaceutical
composition, in combination with a pharmaceutically acceptable vehicle.
These compositions may be administered for example orally, enterally,
parenterally or topically. Preferably, the pharmaceutical composition is
applied topically. By the oral route, the pharmaceutical composition may
be provided in the form of tablets, gelatin capsules, sugar-coated
tablets, syrups, suspensions, solutions, powders, granules, emulsions,
suspensions of microspheres or nanospheres or lipid or polymer vesicles
allowing controlled release. By the parenteral route, the pharmaceutical
composition may be provided in the form of solutions or suspensions for
infusion or injection.

[0063]By the topical route, the pharmaceutical composition is more
particularly useful for the treatment of the skin and the mucous
membranes and may be provided in the form of salves, creams, milks,
ointments, powders, impregnated pads, solutions, gels, sprays, lotions or
suspensions. It may also be provided in the form of suspensions of
microspheres or nanospheres or of lipid or polymer vesicles or of polymer
patches or hydrogels allowing controlled release. This composition for
topical application may be provided in anhydrous form, in aqueous form or
in the form of an emulsion. In a preferred embodiment, the pharmaceutical
composition is provided in the form of a gel, a cream or a lotion.

[0064]The composition may comprise an amount of ELOVL5 modulator ranging
from 0.001 to 10% by weight, in particular from 0.01 to 5% by weight
relative to the total weight of the composition.

[0065]The pharmaceutical composition may additionally contain inert
additives or combinations of these additives, such as:

[0075]FIGS. 1A, 1B and 1C show the expression of ELOVL5 in the sebaceous
gland of the mouse skin and in the mouse preputial gland by in situ
hybridization. FIG. 1A under conventional illumination and under
illumination with a dark background is a photograph of a mouse skin
section subjected to in situ hybridization using an ELOVL5 sense probe
(negative control; animal 44, gonadectomized). FIG. 1B under conventional
illumination and under illumination with a dark background is a
photograph of a mouse skin section subjected to in situ hybridization
with an anti-sense probe, in an intact animal (animal 44). FIG. 1c under
conventional illumination is a photograph of a mouse skin section
subjected to an in situ hybridization with an anti-sense probe, in a
gonadectomized animal (animal 53).

[0076]FIGS. 2A, 2B and 2C show the expression of ELOVL5 in the preputial
gland of mice by in situ hybridization. FIG. 2A is a photograph under
conventional illumination and under illumination with a dark background
of a mouse prepuce section subjected to an in situ hybridization with an
ELOVL5 sense probe (negative control; animal 43). FIG. 2B is a photograph
under conventional illumination and under illumination with a dark
background of a mouse prepuce section subjected to an in situ
hybridization with an anti-sense probe, in an intact animal (animal 43).
FIG. 2C is a photograph under illumination with a dark background of a
mouse prepuce section subjected to an in situ hybridization with an
anti-sense probe, in a gonadectomized animal (animal 53).

[0077]FIGS. 3A and 3B are graphs which show the measurement of the
expression of the ELOVL5 gene in gonadectomized male mice treated with
the vehicle, DHT, DHEA or the combination of DHEA-Flutamide for a period
of 7 days once per day (long-term treatment). The results obtained by the
Affymetrix technique (FIG. 3A) were confirmed by the real-time RT-PCR
technique (FIG. 3B).

[0080]DHEA: gonadectomized mice treated with Dihydroepiandrosterone
(precursor of the steroid hormones; in the preputial glands metabolized
to the active androgen).

[0081]DHEA-Flu: gonadectomized mice treated with a combination of
Dihydroepiandrosterone and Flutamide (antagonists of the androgen
receptor; which block the effects of the DHT and DHEA agonists).

[0082]Level of expression: level of expression of the mRNA.

[0083]In order to further illustrate the present invention and the
advantages thereof, the following specific examples are given, it being
understood that same are intended only as illustrative and in nowise
limitative. In said examples to follow, all parts and percentages are
given by weight, unless otherwise indicated.

EXAMPLES

Experimental Data

Example 1

Expression of ELOVL5 in the Human Sebaceous Gland and in the Human
Epidermis

[0084]Methods:

[0085]Human sebaceous glands were separated from the human epidermis by
treatment with dispase and dissection under a binocular lens. Samples of
total RNA were prepared from the sebaceous glands and from the epidermis.

[0086]The expression of the genes was analyzed on an Affymetrix station
(microfluidic model; hybridization oven; scanner; computer) following the
protocols provided by the company. Briefly, the total RNA isolated from
the tissues is transcribed to cDNA. From the double-stranded cDNA, a cRNA
labeled with biotin is synthesized using T7 polymerase and a precursor
NTP conjugated to biotin. The cRNAs are then fragmented to small sized
fragments. All the molecular biology steps are checked using the Agilent
"Lab on a chip" system in order to confirm the good efficiencies of the
enzymatic reactions. The Affymetrix chip is hybridized with the
biotinylated cRNA, rinsed and then fluorescence labeled using a
fluorophore conjugated to streptavidin. After washings, the chip is
scanned and the results are calculated using the MAS5 software provided
by Affymetrix. An expression value is obtained for each gene as well as
the indication of the significance of the value obtained. The calculation
of the significance of the expression is based on the analysis of the
signals, which are obtained following hybridization of the cRNA of a
given gene with an oligonucleotide that is a perfect match compared with
an oligonucleotide which contains a single mismatch in the central region
of the oligonucleotide (see Table 1).

TABLE-US-00001
TABLE 1
measurement of the expression of ELOVL5 in the epidermis and in the human
sebaceous gland through the use of the Affymetrix chip technology.
Significance of Significance of
Expression Expression the expression* the expression*
Affymetrix Name of in the human in the human in the human in the human
identifier the gene sebaceous gland epidermis sebaceous gland epidermis
208788_at ELOVL5 1295 273 1 1
*Indicator of the significance of the expression of the gene analyzed in
the sample indicated: presence (=1) or absence (=0).

Results

[0087]ELOVL5 is well expressed in both tissues (sebaceous gland,
epidermis). Differential analysis between the expression in the human
sebaceous gland and the human epidermis shows that the slightly higher
expression in the sebaceous gland is not significant compared with the
value observed in the epidermis (Table 1).

Example 2

Expression of ELOVL5 in the Sebaceous Gland Of Mouse Skin by "In Situ
Hybridization"

[0088]Methods:

[0089]Sense and anti-sense probes were prepared from the ELOVL5 gene by
incubation of the linearized gene (2 μg) with 63 μCi of
[35S]UTP (1250 Ci/mmol; NEN, Massachusetts, USA) in the presence of
T7 or T3 RNA polymerase. The in situ hybridization was carried out on a
mouse tissue fixed with formaldehyde and embedded in paraffin. Sections
(4 μm wide) were then deparaffinized in toluene and rehydrated by an
alcohol gradient. After drying, the various sections were incubated in a
prehybridization buffer for two hours. The hybridization was carried out
overnight in a hybridization buffer (prehybridization buffer with 10 mM
DTT and 2×106 cpm RNA/μl 35S-labeled) at 53° C.
The excess probe was removed and the sections were inclined in an LM1
emulsion (Amersham Biosciences, UK) and exposed in the dark at 4°
C. for at least one month. The sections were then developed and
counterstained with hematoxylin and eosin. The hybridization with the
sense probe was used as negative control and only the background was
detected. These probes were incubated with histological sections of mouse
skin or mouse preputial gland. Following incubation in the presence of a
photographic emulsion, the histological structures radioactively labeled
with the probe are visualized (accumulation of silver grains). A specific
signal manifests itself by a positive labeling with the anti-sense probe
(FIG. 1B and FIG. 1c) and the absence of labeling with the sense probe
(FIG. 1A) used as negative control.

Results

[0090]It is observed in FIG. 1A that there is no accumulation of silver
grains (no labeling), which is in agreement with the expectations of the
researchers because it corresponds to the negative control. FIG. 1B shows
a very strong labeling of the sebaceous gland, visible by accumulation of
silver grains. FIG. 1c also shows a labeling of the sebaceous gland.

[0091]ELOVL5 is well expressed in the basal layers of the sebaceous glands
of mouse skin. Fine analysis based on the observation of histological
sections obtained for 4 intact animals and 4 gonadectomized animals does
not indicate strong differences in expression between the sebaceous
glands of the intact animals and the sebaceous glands of the
gonadectomized animals.

Example 3

Expression of ELOVL5 in the Mouse Preputial Gland by In Situ Hybridization

Methods

[0092]The methods used in this example are identical to those of Example
2.

[0093]The mouse preputial glands show a sebocyte type differentiation and
are used as an experimental model of the sebaceous gland.

Results

[0094]FIG. 2A shows no labeling at the level of the preputial gland, which
is in agreement with the expectations of the researchers because it
corresponds to the negative control. FIG. 2B shows a high labeling of the
mouse preputial gland in a normal animal. FIG. 2C shows a very high
labeling of the acini of the preputial gland in a gonadectomized animal.

[0095]ELOVL5 is expressed in (FIG. 2B) the mouse preputial gland. Analysis
of several histological sections from 4 control animals and 4
gonadectomized animals indicates a slightly higher expression in the
preputial glands of the intact animals (FIGS. 2B and 2C).

[0096]In short, these results of in situ hybridization indicate that the
expression of the ELOVL5 enzyme increases under conditions characterized
by a lack of androgenic stimulation (gonadectomized animals).

Example 4

Expression of ELOVL5 in the Mouse Preputial Gland

[0097]The mouse preputial glands show differentiation of the sebocyte type
and are used as an experimental model for a sebaceous gland. They have a
sufficient size to allow isolation of RNA without having recourse to
microdissection technologies.

[0098]Analysis of the expression of ELOVL5 in the mouse preputial glands
was carried out under conditions of deficiencies of steroid hormones (in
particular of androgenic hormones) following a gonadectomy. The
gonadectomized animals were then treated with physiological quantities of
Dihydrotestosterone (DHT) or Dihydroepiandrosterone (DHEA) in order to
restore a physiological level of androgenic hormones, or as a control
experiment with a DHEA-Flutamide combination in which the Flutamide, an
antagonist of the androgen receptors, blocks the effect of DHEA.
Comparison of the gene expression under these experimental conditions
makes it possible to unambiguously identify the modulation or
non-modulation of the gene expression of a gene in question by the
androgenic hormones.

[0099]The gene expression was analyzed using the Affymetrix technology
described above (FIG. 3A) and the results were then confirmed by the
real-time PCR technique (FIG. 3B).

[0100]The real-time PCR was carried out using the protocols provided by
the company Applied Biosystems using the 7900HT Sequence Detection
System. The total RNA isolated from the tissues is transcribed (RT) to
cDNA and the latter is amplified by PCR (Polymerase Chain Reaction). The
progress of the PCR is monitored in real time using fluorescent TaqMan
probes which allow precise quantification of the quantity of mRNA of a
given gene present in the biological sample at the start.

Result

[0101]The mRNA for ELOVL5 is reduced by a chronic treatment for 7 days
with androgens in the preputial gland.

[0103]While the invention has been described in terms of various specific
and preferred embodiments, the skilled artisan will appreciate that
various modifications, substitutions, omissions, and changes may be made
without departing from the spirit thereof. Accordingly, it is intended
that the scope of the present invention be limited solely by the scope of
the following claims, including equivalents thereof.